Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse pluripotent embryocarcinoma cell line P19 after sodium valproate treatment to investigate its mechanism of teratogenicity

ABSTRACT: In the process of evaluating teratogenic properties of xenobiotica, the consumption of laboratory animals is high and costly which makes the development of alternative methods desirable. The pluripotent embryocarcinoma cell line P19, which closely resembles the early stage of an embryo, may be relevant as an in vitro model of teratogenicity. The antiepileptic drug Valproic acid (VPA) is a potent inducer of neural tube defects (ntd:s) in both human and mouse, but its mechanism of teratogenicity is not know. P19 cells were here treated with sodium valproate in a both time and dose dependent matter to profile the gene expression response with Codelink UniSet Mouse 20K I Bioarrays. This profile may reveal biomarkers of ntd:s as well as give mechanistic input of the teratogenicity of VPA.

INSTRUMENT(S): GenePix 4000B [Axon Instruments]

ORGANISM(S): Mus musculus

SUBMITTER: Måns Jergil

PROVIDER: E-MEXP-981 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Prenatal exposure to the anti-seizure drug sodium valproate (VPA) is associated with an increased risk of adverse postnatal neurodevelopmental outcomes, including lowered intellectual ability, autism spectrum disorder and attention-deficit hyperactivity disorder. In this study, we aimed to clarify the molecular mechanisms underpinning the neurodevelopmental consequences of gestational VPA exposure using integrative genomics. First, we assessed the effect of gestational VPA on fetal brain gene expression using a validated rat model of valproate teratogenicity that mimics the human scenario of chronic oral valproate treatment during pregnancy at doses which are therapeutically relevant to the treatment of epilepsy. Two different rat strains were studied - inbred Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a model of genetic generalized epilepsy, and inbred Non-Epileptic Control (NEC) rats. Female rats were fed standard chow or VPA mixed in standard chow for 2 weeks prior to conception and then mated with same-strain males. In the VPA-exposed rats maternal oral treatment was continued throughout pregnancy. Fetuses were extracted via C-section on gestational day 21 (one day prior to birth) and fetal brains were snap frozen and genome-wide gene expression data generated. We found that gestational VPA exposure via chronic maternal oral dosing was associated with substantial drug-induced differential gene expression in the pup brains, including dysregulated splicing, and observed that this occurred in the absence of evidence for significant neuronal gain or loss. The set of genes down-regulated by VPA in the pup brains were significantly enriched for pathways related to neurodevelopment and synaptic function, and significantly enriched for heritability to human intelligence, schizophrenia and bipolar disorder. Our results provide a mechanistic link between chronic fetal VPA exposure and adverse neurodevelopmental outcomes mediated by VPA-induced transcriptional dysregulation.

Project description:The molecular hallmark of the Ewing family of tumors is the presence of balanced chromosomal translocations leading to the formation of chimerical transcription factors (i.e. EWS/FLI1) that play a pivotal role in the pathogenesis of Ewing tumors by deregulating gene expression. We have recently demonstrated that DAX1 (NR0B1), an orphan nuclear receptor which was not previously implicated in cancer, is induced by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors, suggesting that DAX1 is a biologically relevant target of EWS/FLI1-mediated oncogenesis. In this work we demonstrate that DAX1 is a direct transcriptional target of the EWS/FLI1 oncoprotein through its binding to a GGA-rich region in the DAX1 promoter and show that DAX1 is a key player of EWS/FLI1-mediated oncogenesis. DAX1 silencing using an inducible model of RNA interference induces growth arrest in the A673 Ewing cell line and severely impairs its capability to grow in semisolid medium and form tumors in immunodeficient mice. Gene expression profile analysis demonstrated that about ten percent of the genes regulated by EWS/FLI1 in Ewing cells are DAX1 targets, confirming the importance of DAX1 in Ewing oncogenesis. These findings indicate that DAX1 is an important player in the pathogenesis of the Ewing family of tumors, identify new functions for DAX1 as a cell cycle progression regulator and open the possibility to new therapeutic approaches based on DAX1 function interference. A673 cells derived from Ewing sarcoma were genetically enginereed to express specific shRNAs against GFP (control), EWS/FLI1 and DAX1 upon doxycycline stimulation. Three independent clones and a polyclonal population from each enginereed cell were analyzed. Cells were stimulated with doxycycline for 72 hours to induce the expression of the corresponding shRNA and whole gene expression profile performed. Gene expression profile in A673 cells in which EWS/FLI1 or DAX1 were silenced were compared to the control cells.

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Transcription profiling of mouse pluripotent embryocarcinoma cell line P19 after sodium valproate treatment to investigate its mechanism of teratogenicity

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